Cultured Human Skin Fibroblasts and Keratinocytes: Differences in the Regulation of Cholesterol Synthesis

0022-202X/83/ 8102-0125$02.00/ 0 THE JOURNAL OF INVESTIGATIVE DERMATOLOG Y,

Vol. 81 , No.2

81:125-130, 1983

Printed in U. S .A .

Copyright © 1983 by The Willia ms & Wilkins Co.

Cultured Human Skin Fibroblasts and Keratinocytes: Differences in the Regulation of Cholesterol Synthesis MARIA PONEC, Ph.D., LOUIS HAVEKES, Ph.D., JOHANNA KEMPENAAR, AND BERT JAN VERMEER, M.D.

Department of Dermatology, University Hospital, and Gaubius Institute TNO (LH), Leiden, The N etherlands

The regulation of cholesterol synthesis in cultured human skin fibroblasts and keratinocytes was compared, the incorporation of e•c]-acetate or e•c]-octanoate into [l 4 C]-cholesterol being taken as a measure of de novo cholesterol synthesis. The two types of cultured cells differed in the following features of the regulation of cholesterol synthesis: (1) Keratinocytes synthesized 10-fold more cholesterol/mg cell protein. (2) Keratinocytes retained a greater amount of the de novo synthesized cholesterol intracellularly, and fibroblasts released it to a much higher degree into the culture medium. (3) When the extracellular environment was deprived of cholesterol, the intracellular synthesis remained virtually unchanged in keratinocytes but increased markedly in fibroblasts. (4) The low-density lipoproteins (LDL) that enter the cells by receptor-mediated endocytosis and are then degraded in lysosomes, liberate cholesterol, which in turn interferes with the intracellular cholesterol synthesis. The lipoproteins strongly suppress cholesterol synthesis in fibroblasts, but do not have this effect in keratinocytes. (5) When added to the culture medium, nonlipoprotein cholesterol produced no effect on cholesterol synthesis in keratinocytes, whereas fibroblasts showed a marked suppression of this synthesis. The addition of 25-hydroxycholesterol to the culture medium led to a strong suppression of cholesterol synthesis in both fibroblasts and keratinocytes. These findings suggest that in both cell types the 3-hydroxy-3methylglutaryl coenzyme A reductase activity can be suppressed by a sterol delivered to the cell in artificial nonlipoprotein form. (6) The amount of [ 1251]-LDL bound specifically to the cell membrane receptor and particularly the amount internalized and degraded by the cells is much lower in keratinocytes than in fibroblasts, as shown biochemically. In the ultrastructural studies no binding of LDL to keratinocytes was observed. The epidermis, the outermost layer of the skin, is composed of various layers of epidermal cells that are in different stages of differentiation. Two processes occur in the epidermal cells in the basal layer, i.e., proliferation and differentiation. The proliferative activity of some of the cells provides the skin layer with new cells. The remaining cells undergo a differentiation process during which the cells gradually change and ultimately Manuscript received July 1, 1982; accepted for publication December 2, 1982. This work was supported in part by the Centre International de Recherches Dermatologiques, Valbonne France. Reprint requests to: Dr. M. Ponec, Department of Dermatology, University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands. Abbreviations: EGF: epidermal growth factor FCS: fetal calf serum HMGCoA: 3-hydroxy-3-methylglutaryl coenzyme A LDL: low-density lipoprotein LPDS: lipoprotein-deficient newborn calf serum NBCS: newborn calf serum PBS: phosphate-buffered saline

form the outermost layer ofthe epidermis, the stratum corneum [1]. The stratum corneum plays a very important role in the protection of the human body against the loss of physiologically important substances as well as against the infiltration of foreign bodies. It has often been suggested that lipids are important determinants of skin penetrability [2,3]. The lipid fraction of epidermis was found to represent about 5% of the total weight. During the differentiation process, changes in lipid composition occur [3,4]. In the stratum corneum the phospholipid content decreases and that of cholesterol increases with respect to the granular layer [3,4]. A considerable amount of cholesterol was found to be excreted by skin (about 85 mg per day) [5,6]. According to Nicolaides and Rothman [7], this cholesterol originates mainly from the epidermis and only partially from sebaceous glands. The excreted cholesterol is present in 2 forms, i.e., as free cholesterol and esterified cholesterol. Esterification of cholesterol takes place in the epidermis and stratum corneum, but mainly in the surface f1lm [8]. The ratio of the free to the esterified cholesterol varies markedly with the anatomical site, and is inversely related to the density of sebaceous glands [9]. Information has accumulated in the recent literature on the cholesterol uptake in various cells [10]. In most extrahepatic cells, the exogenous cholesterol which is mainly present as cholesteryl esters in low-density lipoproteins (LDL) is taken up by the cells by a receptor-mediated mechanism [10]. By this mechanism LDL is first bound to a specific plasma membrane receptor and then internalized and degraded in lysosomes. The cholesterol liberated from LDL after its degradation has been proved to regulate the intracellular cholesterol synthesis by influencing the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. When the cells are deprived of exogeneous cholesterol, e.g., by incubation in lipoprotein-deficient (LPDS) medium, a marked increase of the endogenous cholesterol synthesis takes place. After the addition of LDL to such cells preincubated in medium supplemented with LPDS, suppression of cholesterol synthesis has been observed in many cell systems (for review, see [10]) . In diseased skin, for example in X-linked ichthyosis, Refsum's disease, and neutral lipid storage disease, alterations occurring in the differentiation process can result in either thinning or thickening of the upper layer of the epidermis [11]. The "abnormal" upper layer may have an abnormal lipid content, and this can be accompanied by an altered cell-cell adhesiveness. It has recently been shown [12] that changes in the cellular cholesterol content have a strong influence on cell-cell or cell-substratum adhesion of Chinese hamster lung and ovary cells and murine L cells. Since the cellular cholesterol content seems to play an important role in the epidermal cell-cell adhesiveness and determines the penetrability of this layer, we decided to study the processes involved in the regulation of the cholesterol synthesis in cultured human epidermal keratinocytes. MATERIALS AND METHODS Cell Culture Fibroblasts: The isolation and culture of human foreskin fibroblasts was performed as described elsewhere [13]. For the experiments, 2 X 5 10 cells were inoculated on a liquid medium composed of 5 ml Ham's

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F 10 medium supplemented with 15% newborn calf serum (NBCS), streptomycin (100 mg/ml), and penicillin (100 U/ml) in Petri dishes (Greiner, 60-mm diameter) and cultured in humidified atmosphere containing 5% CO z. The medium was renewed twice a week, and conflu ency was reached in about 1 week. For the studies concerning the effect of cell density on cholesterol synthesis, the cells were inoculated at various densities from 1.24- 15 x 105 cells per rush and cultured for 2 days. Twenty-four hours before the assays were started the cultures were washed twice with phosphate-buffered saline (PBS) (pH 7.4) and then provided with 5 ml medium supplemented with either 15% NBCS or 15% LPDS. Keratinocytes: The keratinocytes originating from juvenile foreskins were isolated and cultured according to the slightly modified method of Rheinwald and Green [14], as described elsewhere [15]. For these experiments, 3 X 105 keratinocytes were plated together with 8 x 105 feeder cells [gamma-irradiated (3000 R) 3T3 mouse fibroblasts] on 60mm Petri rushes (Greiner) in 5 ml Dulbecco-Vogt medium supplemented with 20% fetal calf serum (FCS), strepto mycin (100 mg/ml), penicillin (100 U/ml), w-G M isoproterenol (Sigma), and 0.4 pg hydrocortisone/ml, and incubation was performed in a humidified atmosphere with 10% C0 2 at 37°C. After 3-4 days, the medium was renewed with similar medium but containing in addition 10 ng epidermal growth factor (EGF) / ml (Sochiba, France). This medium was renewed twice a week, and confluency occurred within 10-14 days after plating. For the studies on the effect of the cell density on cholesterol synthesis, 0.2-3 x 105 keratinocytes and 8 x 105 feeder cells were combined, plated, and cultured for 8-10 days. Twenty-four how·s before the assay, the cells were preconditioned by incubation in Dulbecco-Vogt medium supplemented with either 15% NBCS or 15% LPDS. The only exception was made for nonconfluent cultures; in these cases, 1 day before preconditioning of the cells the remaining 3T3 cells were removed by brief treatment with 0.02% EDTA, after which the cultures were washed twice with PBS and cultmed for 24 h in medium supplemented with 20% FCS. Lipoproteins: Human LDL (d = 1.03-1.05 g/ml) was isolated from plasma of healthy subj ects by differential ultracentrifugation [16]. LPDS (d > 1.215 g/ml) was prepared by ultracentrifugation at density of 1.215 g/ml followed by dialysis against saline. LDL was labeled with 125 1 according to Bilheimer et a l [17].

Measurement of the Incorporation of ["C) -Acetate or [' "'C]-Octanoate into ['''C)-Cholesterol · For conditioning of fibroblasts or keratinocytes, the cells were incubated for 24 h in Ham's F 10 or Dulbecco-Vogt medium, both containing either 15% NBCS or 15% LPDS. To assess the effects of LDL or extracellular cholesterol on the de novo cholesterol synthesis, the cells were preconditioned in medium containing LPDS and reincubated for 6 h in medium supplemented with LDL, cholesterol, or 25-hydroxycholesterol (Steraloids) at concentrations of 0-100 pg cholesterol/ml meilium. Cholesterol and 25-hydroxycholesterol were added in 5 J.!l ethanol per rush. The controls received only 5 J.!l ethanoL For the measmement of de novo cholesterol synthesis, ['''C)-acetate (The Radiochemical Center Amersham, 59 mCi/mmol; 2.5 JLCi/ml medium) at a final concentration of 0.5 mM was added or ['''C]-octanoate (NEN, 25.1 mCi/ mmol, 3 J.!Ci/ml medium) at various concentrations, after which the cells were incubated for 18 h. All incubations were performed at 37°C in humidified atmosphere containing 5% COz (for fibroblasts) or 10% C02 (for keratinocytes). After incubation the medium was removed and cells were washed 3 times with PBS and digested in 1 N NaOH. A sample was taken for protein determination [18] and the amount of ['"C)-cholesterol was then measured in the cell fraction and medium according to a slight modification of Goldstein et al's [19] method. The extracted lipids were separated by thin-layer chromatography using Kieselgel 60 plates (Merck) in seq uental elution, i.e., chloroform:methanol (98:2) followed by chloroform:n-hexane (25:60}. In the first system about two thirds of the plate was deve loped. The Rr value for cholesterol was 0.3. For all samples, a correction was made for recovery by adding an internal standard of [1,3-"H]-(N)-cholesterol (NEN, 53 Ci/mmol) at the beginning of the extraction procedure (3 X 10" dpm/sample). Recovery ranged between 65 and 80%. Binding, Internalization, and Degradation of LDL by Keratinocytes and Fibroblasts Before the assay, the keratinocytes and fibroblasts in dishes 60 mm in diameter were preincubated with LPDS-containing medium to express t he LDL receptor. [ 12"1]-LDL was then added (25 JLg/ml) and the cells were reincubated at 37°C for 3 h. The binding, internalization, and degradation of (' 251]-LDL was measured essentially according to Goldstein and Brown [20]. In short, degradation was measured as free-iodine

trichloroacetic acid-soluble radioactivity in the medium, binding was measured as the amount of radioactivity released from the cells after incubation with heparin, and the measure applied for internalization was the total amount of cell-associated radioactivity still present after incubation with heparin.

Immunoelectron Microscopical Procedures Keratinocytes seeded in a Petri dish were cultured on pieces of Melinex and exposed for 48 h to medium containing 15% (v/v) LPDS. Four incubations were then performed: 2 h at 4°C with LDL (at a concentration of 50 JLg protein/ml medium) , 30 min at 37°C with LDL, 2 hat 4°C without LDL, and 30 min at 37°C without LDL. The next procedure applied has been described in detail [21]. After incubation, the cells were intensively washed with a buffer containing albumin and with PBS, and then prefixed for 10 min at 4°C with 1% paraformaldehyde. This was followed by conventional 2-step indirect immunoperoxidase technique with the use of antihuman apoprotein B 1:20 in the first step and goat antirabbit IgG conjugated with horseradish peroxidase 1:50 (Nordic, Tilburg, The Netherlands) in the second. After the illaminobenzidine H 2 0 2 reaction, the cells were postfixed with 1% Os0 4 in 0.1 M cacodylate buffer (pH 7.4), dehydrated in a graded series of ethanol up to 70%, and embedded in situ. Ultrathin sections unstained or stained with lead hydroxide were examined in a Philips EM 200 electron microscope operated at 80 kV with a 5-JLm objective aperture. The antiserum against human apoprotein B was produced in rabbits by immunization with pme human apoprotein B, as described elsewhere [22]. The antiserum reacted only with low and very low density lipoproteins, as shown in immunoelectrophoresis and double immunodiffusion.

Sampling and Analysis of Data All experiments were performed in duplicate; for each experimental condition at least 6 grids were examined. For the experiment in which keratinocytes were incubated for 2 h at 4°C with LDL, quantitative analyses were performed in duplicate. Material from this experiment was analyzed on 3 grids, each representing a different Epon block. The plasma membrane was photographed at random. An X-Y tablet (MOP AMO 2 Rontron) was used to measure 1 mm of the plasma membrane on positive prints at a final magnification of 20,000. A detailed description of this procedure has been published elsewhere [21]. RESULTS A significant difference was found between cultm·ed human skin fibroblasts and keratinocytes as to the regulation of intracellular cholesterol synthesis.

Cholesterol Synthesis The de novo cholesterol synthesis was measured as the incorporation of [ 14 C)-acetate into ['"C)-cholesterol. In confluent cultures of fibroblasts this synthesis (exp1·essed per mg cellular protein) was significantly lower than in those of keratinocytes. In two experiments (Table I, A and B), fibroblasts and keratinocytes from the same donor were investigated. Preincubation of cells in medium supplemented with LPDS led to a marked increase of cholesterol synthesis by fibroblasts (approximately 6- to 7-fold), whereas keratinocytes did not show this effect. 4 Objections have been raised concerning the use of [' C]acetate incorporation into ['"C)-cholesterol as a measure of cellular cholesterol synthesis [23]. Since the acetate-pool may be different in various cell types and in cells under different culture conditions, the incorporation of ['"C]-octanoate is assumed to be better for this purpose since it is less dependent on the state of cellular metabolism [23]. Therefore, both the 4 ['"C)-acetate and the [ 14 C]-octanoate incorporation into [' C]cholesterol were measured. When various concentrations of ['"C]-octanoate (0.1-0.7 mM) were used and its corporation into ['"C)-cholesterol was measured, the same differences between keratinocytes a nd fibroblasts were found in de novo cholesterol synthesis as with ['"C)-acetate (Table II).

Excretion of Cholesterol into Medium The fraction of the de novo-synthesized cholesterol excreted into the culture medium by fibroblasts during the period (18 h) of labeling with ['"C)-acetate was high (40-55% in medium

CHOLESTEROL SYNTHESIS IN KERATINOCYTES AND FIBROBLASTS

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TABLE I. The effect of preconditioning in LPDS on the cholesterol synth esis in conflu ent cultures of human shin fibr oblasts (F) and heratinocytes (K)

Skin donor

Cell

Cholesterol synthesized (nmol/mg protein) in the presence of: 15%

K

A

F B

K

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F

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15% LPDS

NBCS

13.5 3.8 4.1 1.3 11.5 6.5 3.3 3.7

12.5 0.6 3.6 0.2 11.0 5.4 3.2 0.5

NBCS

15% LPDS

0.9 11 0.4 15 2.0 1.7 1.4 13

6.7 41 3.2 55 6.2 9 8.6 50

F F The confluent cultures of fibroblasts or keratinocytes were preconditioned for 24 h in medium supplemented with either 15% newborn calf serum (NBCS) or 15% lipoprotein-deficient newborn calf serum (LPDS). For the measurement of de novo cholesterol synthesis ["C)acetate (2.5 ILCi/ml medium; 0.5 mM) was added and the cells were further incubated for 18 h. Subsequently, ["C)-cholesterol was measured in both cell layer and medium. TABLE II. The incorporation of ['''Cj-octanoate into [' 4 C] cholesterol in conflu ent cultures of human skin fibrobla sts (F) and heratinocytes (K) incubated in medium containing 15% newborn calf serum

Skin donor I

Cell F K

F

II

K Ill

F K

4 10 dpm/dlah

1.5

Percent cholesterol excreted in to the medium in the presence of: 15%

dpm/ mg Protein 6,400 39,000 3,600 21,000 10,200 100,800

14

Three ILCi/ml medium of [ C}octanoate of end concentration 0.13 mM was added, and the cells were incubated for 18 h. The de novo synthesized ["C)-cholesterol was measured after lipid extraction in both cell layer and medium. The values are means of triplicate estimations and represent the sum of ["C)-cholesterol present in the cell layer and excreted into the medium. supplemented with NBCS, 11-15% in that with LPDS) . In the corresponding experiments with keratinocytes, the excretion of synthesized cholesterol did not exceed 10% in medium containing NBCS, or 2% in medium with LPDS (Table 1). To follow the course of the de novo-synthesized c·c]-cholesterollonger, cells were washed with PBS after the overnight incubation with c ·c]-acetate and then reincubated for 3 days in medium containing 15% NBCS. The results, which ru:e presented in Fig 1, show that the total amount of C4 C)-cholesterol remained unchanged, and this suggests that the c·c]-cholesterol did not undergo any metabolic changes. For fibroblasts it was found that an increasing amount of the cholesterol originally present in the cells was excreted into the culture medium, whereas the studies on keratinocytes showed that more than 90% of the C4 C]-cholesterol remained in the cells.

Effect of Cell Density on the Cholesterol Metabolism When fibroblasts reach confluency, the de novo synthesis of cholesterol decreases sharply. However, for keratinocytes the cholesterol synthesis per mg protein is not influenced by an increase in cell density (Fig 2). In this respect it is noteworthy t hat the protein content of fibroblast cultures at confluency is approximately 400- 500 llg protein per dish , whereas keratinocyte cultures show about 2- 2.5 mg protein per dish. The higher protein content of the latter cells at confluency is due to a larger number of cells (approximately 2.5 times) and a higher protein to DNA ratio (approximately 1.5-2 times). When, after reaching confluency, the keratinocyte cultures were maintained

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FIG 1. The "fate" of the de novo-synthesized cholesterol. The confluent cultures of fibroblasts and keratinocytes were incubated overnight in medium supplemented with 15% NBCS and with ["C)-acetate (5 ILCi/ dish). Thereafter in 2 dishes the amount of the de novo-synthesized cholesterol present in the cells after washing was determined (day 0) . In other dishes, the cells were washed 3 times with PBS (to get rid of the extracellularly present [ 14 C]-acetate) and incubated further for 1-3 days in medium supplemented with 15% NBCS, and the amount of ("C)-cholesterol present in cells and in medium was determined. All determinations were performed in duplicate. for an additional period of 2-3 weeks, a profound differentiation was observed and the increase in the protein content was more pronounced than the increase in the DNA content (Fig 3a). During this prolonged culture of keratinocytes the de novo cholesterol synthesis, expressed either per mg of cell protein or per llg DNA, decreased (Fig 36).

Feedbach Regulation of Cholesterol Synthesis The addition of increasing amounts of LDL or non-lipoprotein cholesterol to confluent fibroblast cultures preincubated in medium supplemented with LPDS led to a marked suppression of the de novo cholesterol synthesis (Fig 4). An 80-90% suppression of cholesterol synthesis was alxeady observed at concentrations lower than 10 llg cholesterol per ml medium. In contrast, under the same conditions the a ddition of LDL or non-lipoprotein cholesterol at concentrations up to even 100 !Lg/ml did not influence cholesterol synthesis in keratinocyte cultures. LDL did not influence cholesterol synthesis in either nonconfluent on confluent cultures (Table III) . In keratinocytes cultured for 2 passages in medium containing LPDS, the addition of LDL (50 llg cholesterol/ml medium) did not lead to changes in the rate of cholesterol synthesis either (results not shown) . The addition of 25-hydroxycholesterol to the culture medium caused a marked suppression of the de novo cholesterol synthesis in both fibroblasts and keratinocytes. Addition of 5 llg 25-hyill·oxycholesterol per ml medium to cells incubated in LPDS led to more than 90% reduction of the cholesterol synthesis. Binding of LDL to Cultured Keratinocytes In most extrahepatic cells, LDL is taken up by a receptormediated mechanism [10]. We were interested in finding out whether the same uptake mechanism is also present in cultured keratinocytes. To this end, we m easured the binding, internalization, and degradation of C251)-LDL in cultured keratinocytes and compared the results with those obtained for fibroblasts.

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Vol. 81, No. 2

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FIG 2. Effect of cell density on cholesterol synthesis. The cells were inoculated at various densities and cultured for the next 2 days (fibroblasts) or 1 week (keratinocytes). The remaining 3T3 cells in keratinocyte cultures were discarded after brief treatment of the dishes with 0.02% EDTA 1 day before preconditioning of the cells in medium supplemented with 15% NBCS. Twenty-foUl· hours before the assay the cells were preconditioned in medium supplemented with 15% NBCS and subsequently incubated overnight in the presence of 0.5 mM ["C)acetate (5 /LCi!dish), after which the ["C)-cholesterol in the cell layer and medium was analyzed. The values shown represent the sum of ["C)-cholesterol present in the cell layer and excreted into the medium. The determinations were performed in triplicate.

The findings in these experiments are summarized in Table IV. The LDL was found to bind with high affinity to the cell surface of keratinocytes, but the amount, and especially the amounts that were internalized and degraded, were much lower than in fibroblasts. In keratinocytes, the amount of LDL that was internalized or degraded did not exceed the amount of LDL that was bound. This is in sharp contrast with the situation in fibroblasts, where within 3 h internalization and degradation exceeded the binding (Table IV). One possible reason for the low binding, uptake, and degradation of [ 1251]-LDL might lie in insufficient removal of lipoproteins from the culture medium during the 24-h preincubation in LPDS-containing medium. To investigate this point, the cells were first incubated for 24 h in LPDS-containing medium and then for 3 h with [ 125 I]-LDL. Next, the cell cultures were divided into 2 portions. In one portion the binding internalization and degradation of [ 1251)-LDL was measured immediately, and the other portion was washed with albumin-containing buffer to free the cell surface of the nonspecifically bound LDL, after which the cells were incubated for 24 h in a medium containing LPDS. After this period, the binding, internalization, and degradation of [ 125 I)-LDL was measured. As can be seen from Table V, in both fibroblasts and keratinocytes the 24-h incubation in LPDS-containing medium was sufficient to deprive the cell surface of spe"cifically bound LDL.

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FIG 3. Effect of prolonged culture of keratinocytes on protein, DNA, and cholesterol synthesis. The keratinocyte cultures, after having reached confluency (8-10 days of cell culture) , were incubated further for the next 2-3 weeks. Medium that was renew -:! twice a week was supplemented with 5% FCS, 0.4 /Lg hydrocortisone/mi. w -" M isoproterenol, and EGF (10 ng/rnl). Twenty-four hours before labeling and DNA/protein analysis, the cells were fed with medium supplemented with 15% NBCS and subsequently incubated overnight in the presence of 0.5 rnM ["C)-acetate (5 /LCi/dish), after which ["C)-cholesterol in the cell layer and medium was analyzed. The cells designed for DNA and protein analysis were washed 3 times with PBS and incubated for 1 h in 1 N NaOH at 37°C. Aliquots were taken for protein analysis [15] and for DNA analysis [33]. All determinations were performed in triplicate. a, Protein/DNA ratio in 3 different cell lines (0 , 6, 0) . b, Cholesterol synth esis (closed symbols, per /Lg DNA; open symbols, per mg protein).

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FIG 4. Effect of LDL, nonlipoprotein cholesterol, and 25-hydroxycholesterol on the intracellular cholesterol synthesis in keratinocyte and fibroblast cultures. The confluent cultures of fibroblasts (0) and keratinocytes (0 ) were first incubated for 24 h in medium supplemented with 15% LPDS and subsequently for 6 h in medium of the same composition but in the presence of increasing amounts of LDL (0, 0), cholesterol (e, •). or 25-hydroxycholesterol (®, [81) . Thereafter, [' 4 C]acetate (5 /LCi/dish) was added, and after the overnight incubation the synthesized [ 14 C]-cholesterol was analyzed in the medium and the cell layer. The values shown represent the sum of the [' 4 C]-cholesterol present in cells a nd medium, and are the mean values of duplicate determinations.

CHOLESTEROL SYNTHESIS IN KERATINOCYTES AND FIBROBLASTS

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TABLE III. The effect of culture conditions and the presence of lowdensity lipoprotein (LDL) on the cholesterol synthesis in nonconfluent and confluent keratinocyte cultures

Culture conditions"

LDL added per ml medium M

Incorporation of ['''CJ-acetate into ['''C)-cholesterol in cultures• (nmol/ng protein)

Confluent Nonconfluent 12.2 13.2 NBCS 13.8 14.4 LPDS 14.6 13.7 50 LPDS "The keratinocytes were incubated for 24 h in medium supplemented with either 15% newborn calf serum (NBCS) or 15% lipoprotein-deficient newborn calf serum (LPDS) . Next, the medium was renewed and either no or 50 1-1g LDL was added per ml and the cells were incubated for the next 6 h. [' 4 C]-Acetate (2.5 i-!Ci/ml medium; 0.5 mM) was added and the cells were incubated for next 18 h. Subsequently, the incorporation of ["C)-acetate into ['"C)-cholesterol was measured in both cell layer and medium. The values are the means of triplicate estimations. • The protein content per dish in noncontluent cultures was 0.9 mg per dish, in confluent cultures 2.9 mg per dish.

129

titative studies were performed in duplicate on cultured keratinocytes exposed for 2 h at 4 oc with LDL. Even when 1 mm of the plasma membrane was studied in 2 of these experiments, no binding of LDL could be observed. In control experiments with fibroblasts, about 21 LDL binding sites were found on 1 mm of the plasma membrane, as seen previously [21]. DISCUSSION

The synthesis of cholesterol by keratinocytes has been found to be surprisingly high when compared with fibroblasts (Tables I, II). Similar results are reported in the literature. Nicolaides and Rothman [7], who used epidermal slices, found a 10-fold higher c·c]-acetate incorporation than with dermal slices. Chemical analysis according to these investigations also showed that forearm epidermis contains 10 times more sterols than does dermis. The regulation of cholesterol synthesis in keratinocytes differs in many aspects from the cholesterol metabolism in other cell types. Keratinocytes showed not only high cholesterol synthesis, but also marginal excretion of newly synthesized cholesterol into the culture medium (up to IO% within 3 days), TABLE IV. Binding, uptake, and degradation of { 125!}-LDL (low· density protein) by cultured human skin heratinocytes and whereas fibroblasts excrete a large proportion of the cholesterol fibroblasts formed (Table I, Fig I). For both cell types the amount of c•c]-cholesterol secreted into the medium was significantly Amount of C" I]-LDL (ng/mg cell protein) lower when the cells were incubated in LPDS. This was probmg Protein/ dish Cell ably due to the absence of an appropriate acceptor for cholesDegraded Bound Internalized terol in the form of lipoproteins in the cultme medium. 0.8 29 15 29 Keratinocyte Furthermore, unlike fibroblasts, keratinocytes showed no 0.9 33 29 31 change in the rate of cholesterol synthesis with increasing cell 2.0 5 12 9 density, while a decrease in cholesterol synthesis with increasing 3.0 2 3 0 cell density has been demonstrated for various cell types [247 0 3.5 5 26]. This observation has been explained by the finding that in 8.0 5 13 3 high-density cell cultures, where the rate of cell proliferation 0.1 240 595 Fibroblast decreases, less cholesterol is needed for the assemblage of new 200 0.2 500 membranes. However, the growth pattern of keratinocytes dif0.3 40 196 360 fers from that of many cell types which show decreasing cho30 156 220 0.5 lesterol synthesis with increasing cell density. In sparse fibroThe cell cultures after 24-h preincubation in medium supplemented blast cultures the distance between individual cells is relatively with 15% LPDS were further incubated for 3 h at 37°C in the presence large, and with increasing cell density this distance decreases. 125 of ( 1251]-LDL (25 ~/ml) and thereafter the amount of ( I]-LDL bound, In confluent cultures the cells are in direct contact with each internalized, and degraded was determined as described in Materials other and contact inhibition can occur. Sparse keratinocyte and Methods. For determination of nonspecifically bound LDL, the cultures, on the contrary, show small colonies originating from cells were incubated with [ 1251]-LDL in the presence or absence of an 1 or few cells. Except in the peripheral regions of these colonies, excess of nonlabeled LDL (150 1-1g/ml). the keratinocytes are in direct contact with each other throughout the culture period. Between the colonies there is "empty" TABLE V. The effect of "delipidation" on the low-density lipoprotein space which becomes smaller with increasing cell density and (LDL) binding eventually disappears. Cell proliferation is accompanied by After a further differentiation of some of the cells. In keratinocyte cultures in 24-h incubation Amount of [125 !]-LDL After 3 h Cell the early stages of growth, the central part of the colonies with (ng/ mg cell protein) LPDS medium usually shows a multilayer. This suggests that the need for cholesterol remains high in these confluent keratinocyte cul2 Fibroblast Binding 66 75 tures, because some of the cells are always proliferating and Internalization 335 213 Degradation 417 others differentiating. When keratinocytes are kept in culture only a few weeks after confluency has been reached, the amount 2 Keratinocyte Binding 33 of cholesterol synthesized, as expressed per cell protein or per 7 Internalization 26 DNA content, decreases (Fig 3). This suggests that the differ13 Degradation 29 entiating cells require less cholesterol than do proliferating The cells were preincubated 24 h in lipoprotein-deficient newborn cells. calf serum (LPDS) containing medium, and the binding, internalization, In cultured human fibroblasts and other cultured mammalian and degradation of [ 1251]-LDL was measured after 3 h of incubation with [ 1251]-LDL (25 ~/ml) in one part of the cell culture dishes. The cells, the presence of LDL in the growth medium leads to a other culture dishes were washed with albumin-containing buffer (0.2%) sharp decrease in endogenous cholesterol synthesis [10], and incubated for the next 24 h in LPDS-containing medium, after whereas in keratinocytes the regulation of this synthesis is which the amount of [' 251]-LDL that remained bound and the amount apparently unaffected by serum lipoproteins. Furthermore, the addition of LDL or exogenous cholesterol to culture medium that became internalized and degraded during these 24 h was measured. did not reduce the cholesterol synthesis (Fig 4) , even when the cells were fn·st cultured for a long time in LPDS-containing Ultrastructural Studies medium (results not shown). In all other cell systems the effect When keratinocytes were incubated with LDL for . 2 h at 4 °C . of LDL on cholesterol synthesis was only weak or absent when the LDL uptake was either partially or totally defective, as in or for 30 min at 37°C, no specific binding of LDL to the cell the case of the fibroblasts of patients with homozygous familial surface of cultured keratinocytes occurred. To support the morphologic observation that no LDL binding occrn-red, quanhypercholesterolemia [10], mutant cells of Chinese hamster

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ovary cells [27], or human epitheloid A-431 cells [28], or when the intracellular degradation of LDL was disturbed, as for instance in fibroblasts of patients with lysosomal cholesterolesterase deficiency [29). In all of these conditions, exogeneously added non-lipoprotein cholesterol suppressed cholesterol production by bypassing the LDL receptor [10). It should be kept in mind, however, that a strong suppression of the de novo cholesterol synthesis in keratinocytes was already seen after the addition of small amounts (less than 5 1-Lg/ml) of 25-hydroxycholesterol. This finding suggests the presence of the same kind of regulatory mechanism of HMGCoA reductase activity as has been found in other cell systems. The absence of this regulatory mechanism in the presence of non-lipoprotein cholesterol probably is due to the accumulation of cholesterol in the plasma membrane and the very low diffusion rate of cholesterol from the membrane into the cell. The more polar 25hydroxycholesterol penetrates cells more easily and can therefore affect the cholesterol synthesis. In fibroblasts, too, the suppression of ["C)-cholesterol synthesis was achieved at a lower concentration of 25-hydroxycholesterol, which is in a good agreement with data of Brown and Goldstein [30]. In contrast to most cell types discussed in this context in the literature, the amount of LDL bound and, especially, metabolized by keratinocytes was strikingly low (Table IV) . Low rates of internalization and degradation of LDL were also reported recently for epidermoid cervical cancer cells [31), which suggests that the LDL metabolism of differentiating epitheloid cells may be defective. Some similarities seem to exist between keratinocytes and other components of the skin-the hair root, where the rate of cholesterol synthesis was found to be much higher than that in fibroblasts and to be uninfluenced by the presence of LDL [32]. From these data we conclude that the regulation of cholesterol synthesis in cultured keratinocytes is controlled by a unique mechanism that differs from the one found in most of the other cell types. It is of practical importance to find a way to manipulate the cholesterol level in these cells and thus to change the properties of epidermis and especially of stratum corneum by influencing cell-cell adhesion. We wish to thank Marian Wijsman and Hans van der Meer for technical assistance.

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